The present invention relates to a novel reagent for detecting non-A, non-B viral hepatitis NANBH and to a process for diagnosing this type of hepatitis by an immunoenzymatic method.
Non-A, non-B hepatitis is defined as being viral infectious hepatitis, which cannot be attributed to hepatitis A virus (HAV), or to hepatitis B virus (HBV), or to other known human hepatotropic viruses, such as cytomegalovirus, Epstein-Barr virus or yellow fever virus. Non-A, non-B hepatitis seems to represent a group of diseases due to several different viruses.
In Western Europe and North America, 95% of transfusion hepatitis cases are due to infectious agents which are transmitted parenterally in the same way as hepatitis B, as reported by E. TABOR [Lancet, 1985, i: 743-745] and D.G. Wong et al. [Lancet, 1980, ii: 876-879].
Recent epidemiological data have demonstrated the existence of forms of NANBH which are transmitted like hepatitis A, i.e. by the fecal-oral route and virtually never by transfusion. Several epidemics of this type have been reported (M. S. Balayan et al.; Intervirology, 1983, 20: 23-31 - E. H. Belabbes et al.; J. Med. Virol., 1985, 16: 257-263 - M. A. Kane et al.; JAMA, 1984, 252: 3140-3145 - M. S. Khuroo; Am. J. Med., 1980, 68: 818-824). These epidemics are due to the water supply, with a fecal-oral mode of transmission. Viral particles of 27 nm have been observed by electron microscopy in samples obtained from patients' feces (M. S. Balayan et al.; Intervirology, 1983, 20:23-31 - M. A. Kane et al.; JAMA, 1984, 252: 3140-3145).
Non-A, non-B hepatitis viruses are known to cause serious liver diseases. Such as cirrhosis of the liver, non-established malignant hepatoma and the like, so identification of these viruses and detection of the antigen-antibody system associated with these viruses is of the greatest importance in effecting an early diagnosis and rapidly adopting a suitable prophylaxis.
The diagnostic reagents and methods proposed hitherto essentially involve detection of the antigen associated with a non-A, non-B hepatitis virus transmitted during blood transfusions.
Thus, SHIRASHI et al. [THE LANCET, 21 Oct. 1978, p. 853-856] detected an antigen-antibody system, by counterelectrophoresis, in humans suffering from non-A, non-B hepatitis following a transfusion. PCT international application published under number 80/02598, filed on 20 May 1980 and claiming US priority of application Ser. No. 040,921 of 21 May 1979, applied the work of SHIRASHI et al., claiming an immunological test for detecting an antigen associated with non-A, non-B hepatitis (NANBH), in which a sample of serum from a supposedly affected mammal is reacted with an antibody originating from the serum of a donor mammal known to be carrying a NANBH infection, and the presence of the NANBH antigen is detected either indirectly via the immunoprecipitin formed by counterelectrophoresis, or by direct methods, such as radioimmunoassay, diffusion in gelose gel, passive hemagglutination, agglutination on latex, complement fixation or the ELISA test.
Furthermore, PCT international application no. WO 82/00205 describes a viral particle of 50 to 60 nm in diameter which possesses a nucleus of about 40 nm in diameter and is said to be the agent causing non-A, non-B hepatitis. Four samples containing this viral particle were deposited in the American Type Culture Collection under the numbers VR-2011, VR-2012, VR-2013 and VR-2014.
The particles described can be radiolabeled for use in a detection test.
Other authors have attempted to isolate the antigen associated with NANBH and to use this isolated antigen to prepare a reagent for diagnosing the infections caused by the virus of NANB viral hepatitis. In particular, French Patent 81 06385 of 20 Mar. 1981, in the name of TREPO, describes the isolation of a novel NANBH antigen by ultracentrifugation of sera or liver extracts, treatment of the centrifugation residue with a non-ionic detergent, and then fractionation in order to isolate the fractions containing purified NANBc antigen (this being followed, if appropriate, by treatment with an ionic detergent in order to isolate the AgNANBe). According to an alternative proposal corresponding to a variant in the said French patent, after any viral particles present have been removed by ultracentrifugation, as above, the gamma-globulins are removed, the AgNANBe is concentrated by precipitation, the concentrated fractions containing the AgNANBe are chromatographed on a support to which heparin is fixed, and the AgNANBe is collected in the eluates. The diagnostic reagents according to the TREPO French patent comprise a solid support to which at least one NANBc, NANBe or NANBs antigen and/or at least one anti-NANBc, anti-NANBe or anti-NANBs specific antibody are fixed; in the particular case of the diagnostic reagent whose support carries anti-(human gamma-globulin) antibodies, the antigen is separate from the support and can only be fixed via the corresponding antibodies in the serum to be tested, if they are present.
Similarly, the European Patent Application published under the number 66 296, in the name of EISAI, filed on 2 June 1982 and claiming a Japanese priority of 2 June 1981, describes the isolation of an antigen associated with NANB hepatitis by purification from liver taken by autopsy from patients who have died of NANB hepatitis, by conventional methods of protein fractionation, including ultracentrifugation, and defines this antigen by its physicochemical properties; the antigen according to the said European patent application is conjugated with a substance which may consist of particles of substrate suitable for passive hemagglutination, such as sheep erythrocytes, tracer isotopes for radioimmunoassays, or enzymes suitable for carrying out the ELISA test.
To date, however, no means seem to have been proposed which make it possible to identify an antigenantibody system associated with an epidemic form of NANB hepatitis.